NEUROBIOLOGY OF BRAIN ARGININE DECARBOXYLASE

脑精氨酸脱羧酶的神经生物学

• 批准号: 6557992
• 负责人: SOUNDAR REGUNATHAN
• 金额: $ 13.45万
• 依托单位: UNIVERSITY OF MISSISSIPPI MED CTR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-20 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6557992
• 关键词: amines; aminoacid metabolism; arginine; brain metabolism; decarboxylases; enzyme activity; enzyme mechanism; enzyme structure; genetic library; inflammation; laboratory rat; neurochemistry; neurotransmitters; nitric oxide synthase; protein localization; protein purification

In 1994, we isolated agmatine, an amine synthesized from arginine by arginine decarboxylase (ADC), which bound to alpha2 adrenergic and imidazoline receptors of all subclasses, from cow brain. The discovery was a surprise, as neither agmatine nor ADC, prevalent in bacteria, plants and parasites, had been detected in mammals. Following our initial discovery, a number of other studies confirmed the presence of agmatine and ADC in mammalian tissues. Although ADC had been extensively characterized, purified and cloned from bacteria and plants, it was never studied in mammalian systems. The known properties of mammalian (rat brain) ADC clearly distinguishes it from other isoforms and mammalian ornithine decarboxylase, a closely related enzyme. Further studies on the molecular properties, localization and regulation of ADC in mammalian brain is critical in understanding the neurobiology of agmatine. This Proposal will test the hypothesis that mammalian ADC is a novel enzyme with unique primary structure, localized in selective neuronal cells and regulating the production of agmatine, a putative neurotransmitter/neuromodulator. Study 1 will characterize rat liver/brain ADC in parallel with rat liver ODC with respect to subcellular localization, reaction properties, inhibition profiles, and regulation. The proof that mammalian ADC is a novel, and heretofore unrecognized, enzyme will be to establish its primary structure by molecular cloning. We will clone mammalian ADC by two approaches. As several isoforms of non- mammalian ADC have been cloned, mammalian ADC will be cloned by homology screening by PCR to obtain possible partial clones and to use them to screen cDNA libraries to obtain the full length cDNA clone. As an alternate approach, we will purify ADC from rat liver, obtain peptide sequences, and design oligoprobes based on these sequences to be used in PCR based, or cDNA library, screening. Study 2 Will test the hypothesis that ADC may be a glial enzyme, and that its product, agmatine, is released into the extracellular space and taken up into, and stored in, neurons. We will investigate the localization of ADC and agmatine in cultured neuronal cells and whole rat brain by biochemical and immunocytochemical methods to verify this hypothesis. Study 3 will test the hypothesis that activity of ADC is regulated by inflammatory stimuli in glial cells. We will investigate the regulation of the expression of ADC in cultured astrocytes and macrophages by bacterial toxin, LPS, and cytokines. Comparing the regulation of ADC with NOS-2, we will be able to understand how arginine metabolism is regulated in concert with agmatine or nitric oxide synthesis. At the completion of this project, we will be able to demonstrate that mammalian ADC is a novel enzyme with unique sequence, and localization and regulatory properties, thereby providing valuable information and tools to further understand the biological role of agmatine in mammalian brain.

1994年，我们从牛脑中分离到与α2肾上腺素能和咪唑啉受体结合的精氨酸脱羧酶(ADC)催化精氨酸合成的胺。这一发现令人惊讶，因为在细菌、植物和寄生虫中普遍存在的胍丁胺和ADC都没有在哺乳动物中检测到。在我们最初的发现之后，许多其他研究证实了在哺乳动物组织中存在胍丁胺和ADC。虽然ADC已经从细菌和植物中得到了广泛的鉴定、纯化和克隆，但在哺乳动物系统中从未进行过研究。哺乳动物(大鼠脑)ADC的已知性质明显区别于其他同工型和哺乳动物鸟氨酸脱羧酶，鸟氨酸脱羧酶是一种密切相关的酶。进一步研究ADC的分子性质、在哺乳动物脑中的定位和调控对于理解胍丁胺的神经生物学意义重大。这一提议将检验一种假说，即哺乳动物ADC是一种具有独特一级结构的新型酶，定位于选择性神经细胞中，调节可能的神经递质/神经调节剂胍丁胺的产生。研究1将描述大鼠肝/脑ADC与大鼠肝ODC在亚细胞定位、反应特性、抑制谱和调节方面的平行特征。哺乳动物ADC是一种新的、迄今未被认识的酶的证据将通过分子克隆来建立其一级结构。我们将通过两种方法克隆哺乳动物ADC。随着多种非哺乳动物ADC亚型的克隆完成，本论文将通过对哺乳动物ADC进行同源性筛选，获得可能的部分克隆，并利用这些克隆对文库进行筛选，获得全长的cDNA克隆。作为另一种方法，我们将从大鼠肝脏中纯化ADC，获得多肽序列，并根据这些序列设计寡核苷酸探针，用于基于PCR的或cDNA文库的筛选。研究2将检验这样一种假设，即ADC可能是一种胶质酶，其产物胍丁胺被释放到细胞外空间，并被吸收和储存在神经元中。我们将通过生化和免疫细胞化学方法研究ADC和胍丁胺在培养的神经细胞和整个大鼠脑中的定位，以验证这一假说。研究3将验证ADC活性受神经胶质细胞炎症刺激调节的假设。我们将研究细菌毒素、内毒素和细胞因子对培养的星形胶质细胞和巨噬细胞中ADC表达的调节。比较ADC和NOS-2的调节，我们将能够理解精氨酸代谢是如何与胍丁胺或一氧化氮合成一起调节的。在本项目完成后，我们将能够证明哺乳动物ADC是一种具有独特序列、定位和调节特性的新型酶，从而为进一步了解胍丁胺在哺乳动物脑中的生物学作用提供有价值的信息和工具。